GSM6932569: TGFB_LPS, replicate 4, bulk ATACseq; Mus musculus; ATAC-seq
Experiment was performed by Zymo Reserach Corp. Add how you froze the cells / pelleted them. Cells were thawed in a 37°C water bath and then quickly transferred to 9 mL of warm RPMI-1640 with 15% FBS and 1% P/S. The cells were centrifuged at 300 x g for 5 minutes at 25°C. The supernatant was discarded, and the cells were resuspended in 1 mL complete RPMI-1640. 10 μL of the cell suspension was mixed with 10 μL 0.4% Trypan Blue. Cells were counted, and viability was determined using a Countess II (Invitrogen). Both sides of the counting chamber were used, and the numbers reported above are an average of both reads. 300,000 cells were aliquoted to a new tube and centrifuged at 300 x g for 5 minutes at room temperature. The supernatant was removed, and the pellet was resuspended in 150 μL of room temperature ATAC-S Buffer. 50,000 cells were aliquoted to new tubes. An equal volume of cold 2x ATAC Lysis Buffer was added to each aliquot of cells and incubated on ice for 3 minutes, at which point cold ATAC Dilution Buffer was added. These mixtures were centrifuged at 1,000 x g for 10 minutes at 4°C. Nuclei were resuspended in cold Tagmentation Buffer containing the tagmentation enzyme. The tagmentation reaction was carried out at 37°C shaking at 1,000 rpm for 30 minutes. The tagmentation reaction was cleaned up with the DNA Clean & Concentrator Kit-5 (Zymo Research Corp.) and eluted with 21 μL DNA Elution Buffer. The ATAC Seq libraries were prepared in 50 μL PCR reactions consisting of 20 μL of the cleaned up tagmentation products, 2x PCR Master Mix, and 2.5 μM of forward and reverse index primers. The PCR protocol was as follows: 72°C for 5 minutes,  98°C for 30 seconds, 11 cycles of: o 98°C for 10 seconds, 63°C for 30 seconds, 72°C for 60 seconds, Hold at 4°C. The PCR products were cleaned up using the DNA Clean & Concentrator Kit-5 (Zymo Research Corp.) and eluted with 21 μL DNA Elution Buffer. Library QC was performed by analyzing the cleaned-up library via TapeStation (Agilent Technologies). Libraries were quantified using KAPA Library Quantification (Roche) and pooled together for sequencing 150 bp paired-end on the Illumina NovaSeq platform.